HIV encodes a number of accessory genes not commonly found in other retroviruses. The purpose of this project is to investigate the biological and biochemical functions of the HIV accessory proteins Vif and Vpu and to understand their precise role in virus replication. One of our goals is to characterize cellular factors involved in Vif or Vpu function. From our studies on Vpu we expect to gain insights into general principles of protein degradation from the ER and into mechanisms involving late stages of virus production, in particular the involvement of ion channels in the secretory pathway. From our studies on Vif we not only expect to gain insights into the function of this viral factor but we expect to learn more about the role of the cytoskeleton in virus replication. Finally, we hope our studies will provide a basis for the assessment of viral proteins as potential antiviral targets. The vpu gene is unique to HIV-1 and encodes a small integral membrane protein. Vpu regulates virus release from the cell surface and degradation of CD4 in the endoplasmic reticulum. These two biological activities of Vpu are based on two independent and distinct molecular mechanisms that can be attributed to separable structural domains of Vpu. Vpu-regulated virus release is sensitive to changes in the transmembrane (TM) domain of Vpu and is correlated with an ion channel activity of Vpu. CD4 degradation, on the other hand, involves a direct interaction of the Vpu and CD4 cytoplasmic domains. We have now established that Vpu-mediated CD4 degradation involves the ubiquitin-dependent proteasome pathway (Ref 1) and requires an interaction with a novel cellular protein, h-bTrCP (Ref 2). H- bTrCP was found to engage in ternary complexes with Vpu and CD4 - requiring phosphorylation of Vpu ? and represents a direct link to Skp1p, a known component of E3 ubiquitin ligase complexes. HIV-2, which is very closely related to HIV-1, lacks a vpu gene. However, we recently found that in the absence of the HIV-2 Env glycoprotein, Vpu can enhance HIV-2 virus release. In the presence of HIV-2 Env, efficient virus release is observed which was not further enhanced by Vpu. We subsequently observed that some, but not all, of the HIV-2 Env proteins encode an activity that regulates virus particle release, in a Vpu-like manner. We performed a series of experiments to identify domains in HIV-2 Env that are critical for its Vpu-like function. We found that, at least in the case of HIV-2ROD Env, the cytoplasmic domain of Env is not required for this activity. Rather, change of a single amino acid in the ectodomain of an inactive isolate was sufficient to activate its Vpu-like activity. Our data suggest that regulating the Vpu-like activity in HIV-2 Env involves changes in its potential to form homo-oligomers. Vif is a 23-kDa basic protein, which has an important function in regulating infectivity of progeny virions. The biochemical mechanism of Vif function is obscure. We analyzed the role of Vif by studying its subcellular distribution by cell fractionation as well as confocal microscopy. We found that a substantial portion of intracellular Vif protein is associated with the cytoskeleton, specifically intermediate filaments. The association of Vif with intermediate filaments is specific and can result in the reorganization of the cytoskeletal network. In addition, we found that Vif is incorporated into virus particles where it associates with the core of virions. Mutational analysis of Vif suggests that a domain located near the N- terminus of Vif be involved in the interaction with vimentin. The interaction of Vif with vimentin results in the partial or complete reorganization of vimentin and, in extreme cases, culminates in the perinuclear aggregation of vimentin and Vif. Vif-dependent collapse of vimentin is cell-cycle dependent and occurs during late S to G2/M phase.